Project Summary/Abstract Precise organization of neuronal connections is crucial for processing information. A major challenge in neuroscience is to understand how these connections are properly established, and how errors in this wiring process can lead to psychiatric disorders like autism or schizophrenia. During development, neurons extend axons that are guided along defined paths by attractive and repulsive cues to reach their brain target. In addition to this guidance process, mechanisms involving pruning or degeneration correct axons that have deviated from the right path, thereby ensuring accuracy of circuit formation. Several factors regulate axon guidance, but the combined information they provide is not sufficient to sculpt the entire neuronal network. Heparan sulfate proteoglycans (HSPGs) are cell-surface and extracellular core proteins with attached heparan sulfate (HS) sugar chains that play crucial roles in axon pathfinding, and are essential for triggering the selective degeneration of misguided axons. How HSPGs control these different processes remains however a mystery. HS chains undergo many modifications, especially sulfations, that confer on them a unique diversity and structural complexity. These modifications have been proposed to generate a complex ?sugar code? orchestrating the formation of axonal connections by regulating most factors essential for brain wiring. While appealing, this ?sugar code? hypothesis is still theoretical and has not been tested in vertebrates. Moreover, the contribution of the core proteins carrying these specific HS patterns is not known. Using the zebrafish retinotectal system as an in vivo model, I have discovered that three distinct HSPGs (SDC2, GPC1a and GPC1b) regulate different steps of retinal axon guidance. In this project, I propose to dissect out how these core proteins and HS chain modifications regulate axon navigation using a unique combination of in vivo approaches, genetics and HS biochemistry. The first aim of this proposal will confirm the novel functions of SDC2, GPC1a and GPC1b, and investigate their mode of action at a cellular and molecular level. It will determine how their absence affects axon behavior and leads to guidance errors, where they act, whether their HS chains are required for their function, and what are the factors they interact with. The second aim of this project will determine the contribution of specific HS structural motifs for axon pathfinding and degeneration. It will test the roles of the 6-O and 3-O sulfotransferases that modify HS chains with sulfations at specific positions, and examine how synthetic HS oligosaccharides with defined sulfation motifs and sizes regulate retinal axon navigation, both in vitro in culture systems and in vivo in the developing embryo. By studying the roles of both core proteins and HS fine structure, the proposed studies give a unique opportunity to test whether a sugar code orchestrates brain wiring during development. It might also provide new lines of research for future development of therapeutic and regenerative strategies in the context of neurological disorders.